Last year, it was shown that Pax-6 binds to and activates numerous crystallin promoters. This year, additional Pax-6 binding sites in crystallin promoters were identified and evidence was obtained indicating that this transcription factor represses activity of the chicken beta-B1 and beta-A3/A1-crystallin promoters. Cotransfection experiments demonstrated that these beta-crystallin promoters can be activated by another homeodomain transcription factor prevalent in the lens that was cloned last year, Prox-1. A Prox-1 consensus DNA-binding site was provisionally established, and cDNA and gene analyses showed that alternatively spliced forms of Prox-1 are found in different tissues. The cDNA sequence of squid Pax-6 was completed, and the encoded protein was shown to have highly conserved paired and homeodomains with Pax-6 of vertebrates and to be able to induce ectopic eyes in Drosophila. In situ hybridization experiments demonstrated that Pax-6 is expressed in the arms, eyes, olfactory organ, and brain of squid embryos, consistent with its universal role in the evolution of the visual and olfactory systems. A jellyfish Pax gene fragment encoding a paired and small homeodomain related to Pax-2, 5, and 8 was cloned. Evidence was obtained that the much greater lens activity of the chicken delta-1 than delta-2-crystallin gene involves putative silencers in the intergenic spacer and greater responsiveness of the delta-1 than the delta-2 enhancer to Pax-6, Sox-2, and RAR-beta. Retinoic acid receptors were also shown to activate both lens-specific elements of the mouse alpha-B-crystallin promoter in retinoic acid-treated transfected cells. Thus, both Pax-6 and retinoic acid receptors appear to have general roles in crystallin gene expression in the vertebrate lens. As shown earlier for alpha-A, alpha-B-crystallin promoter activity in the lens was shown to be independent of its proximal and distal TATA sequences in transgenic mice. Heat induction of alpha-B promoter activity was also shown to be independent of the TATA sequences. Alpha-B-crystallin was shown to be stress inducible in trabecular meshwork cells, suggesting a possible role for this protein in glaucoma. The major mammalian beta-crystallin, beta-B2, was shown to be autophosphorylated on a different serine than that shown by others to be phosphorylated by a cAMP-dependent process. A similar situation was revealed by us last year for the alpha-crystallins. Although speculative, the findings raise the possibility that these crystallins have metabolic as well as structural roles. Finally, cloned oligodendrocyte cell lines producing alpha-B-crystallin were derived from transgenic mice carrying a gamma-interferon-inducible, temperature-sensitive SV40 T antigen; it is anticipated that these will be extremely useful for studying alpha-B-crystallin promoter activity in brain cells.